ES2283563T3 - PROCEDURE THAT IMPROVES THE REMOVAL OF PROTEASA INHIBITORS. - Google Patents
PROCEDURE THAT IMPROVES THE REMOVAL OF PROTEASA INHIBITORS. Download PDFInfo
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- ES2283563T3 ES2283563T3 ES02737587T ES02737587T ES2283563T3 ES 2283563 T3 ES2283563 T3 ES 2283563T3 ES 02737587 T ES02737587 T ES 02737587T ES 02737587 T ES02737587 T ES 02737587T ES 2283563 T3 ES2283563 T3 ES 2283563T3
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- 238000000034 method Methods 0.000 title claims abstract description 30
- 239000003112 inhibitor Substances 0.000 title claims description 13
- 238000000605 extraction Methods 0.000 claims abstract description 52
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 49
- 235000002639 sodium chloride Nutrition 0.000 claims abstract description 36
- 235000002595 Solanum tuberosum Nutrition 0.000 claims abstract description 35
- 244000061456 Solanum tuberosum Species 0.000 claims abstract description 35
- 239000011780 sodium chloride Substances 0.000 claims abstract description 26
- 239000000137 peptide hydrolase inhibitor Substances 0.000 claims abstract description 22
- 239000000463 material Substances 0.000 claims abstract description 11
- 239000002245 particle Substances 0.000 claims abstract description 11
- 239000000725 suspension Substances 0.000 claims abstract description 11
- 150000003839 salts Chemical class 0.000 claims abstract description 10
- 241000196324 Embryophyta Species 0.000 claims abstract description 9
- 150000007524 organic acids Chemical class 0.000 claims abstract description 8
- 238000001914 filtration Methods 0.000 claims abstract description 6
- 239000007788 liquid Substances 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000007787 solid Substances 0.000 claims abstract description 5
- 238000005119 centrifugation Methods 0.000 claims abstract description 4
- 238000005352 clarification Methods 0.000 claims abstract description 3
- 238000001035 drying Methods 0.000 claims abstract description 3
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 38
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 19
- 235000019253 formic acid Nutrition 0.000 claims description 19
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 11
- 239000002253 acid Substances 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- 108091005804 Peptidases Proteins 0.000 claims description 6
- 102000035195 Peptidases Human genes 0.000 claims description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 5
- 235000019833 protease Nutrition 0.000 claims description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 4
- 239000011668 ascorbic acid Substances 0.000 claims description 2
- 235000010323 ascorbic acid Nutrition 0.000 claims 1
- 229960005070 ascorbic acid Drugs 0.000 claims 1
- 235000013311 vegetables Nutrition 0.000 claims 1
- 238000005507 spraying Methods 0.000 abstract description 2
- 101000622007 Crotalus adamanteus Snake venom metalloproteinase adamalysin-2 Proteins 0.000 description 43
- 239000000243 solution Substances 0.000 description 37
- 102000004169 proteins and genes Human genes 0.000 description 15
- 108090000623 proteins and genes Proteins 0.000 description 15
- 235000012015 potatoes Nutrition 0.000 description 13
- 238000010438 heat treatment Methods 0.000 description 8
- 238000002955 isolation Methods 0.000 description 7
- 239000002904 solvent Substances 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 239000012535 impurity Substances 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 102000005367 Carboxypeptidases Human genes 0.000 description 4
- 108010006303 Carboxypeptidases Proteins 0.000 description 4
- 229920005654 Sephadex Polymers 0.000 description 4
- 239000012507 Sephadex™ Substances 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 108091006086 inhibitor proteins Proteins 0.000 description 4
- 238000005457 optimization Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 229940122644 Chymotrypsin inhibitor Drugs 0.000 description 3
- 101710137926 Chymotrypsin inhibitor Proteins 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
- 229940019748 antifibrinolytic proteinase inhibitors Drugs 0.000 description 3
- 239000003541 chymotrypsin inhibitor Substances 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000004007 reversed phase HPLC Methods 0.000 description 3
- 230000036186 satiety Effects 0.000 description 3
- 235000019627 satiety Nutrition 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- 101001044900 Solanum tuberosum Proteinase inhibitor 1 Proteins 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 235000012054 meals Nutrition 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- PXRKCOCTEMYUEG-UHFFFAOYSA-N 5-aminoisoindole-1,3-dione Chemical compound NC1=CC=C2C(=O)NC(=O)C2=C1 PXRKCOCTEMYUEG-UHFFFAOYSA-N 0.000 description 1
- 102100034613 Annexin A2 Human genes 0.000 description 1
- 229940121981 Carboxypeptidase inhibitor Drugs 0.000 description 1
- 101710127041 Carboxypeptidase inhibitor Proteins 0.000 description 1
- 102100025841 Cholecystokinin Human genes 0.000 description 1
- 101800001982 Cholecystokinin Proteins 0.000 description 1
- 101710140999 Metallocarboxypeptidase inhibitor Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102000002067 Protein Subunits Human genes 0.000 description 1
- 108010001267 Protein Subunits Proteins 0.000 description 1
- 101000986540 Solanum tuberosum Patatin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 229940122618 Trypsin inhibitor Drugs 0.000 description 1
- 101710162629 Trypsin inhibitor Proteins 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000000636 anti-proteolytic effect Effects 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229940107137 cholecystokinin Drugs 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 206010061428 decreased appetite Diseases 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000003880 negative regulation of appetite Effects 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- JVBXVOWTABLYPX-UHFFFAOYSA-L sodium dithionite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])=O JVBXVOWTABLYPX-UHFFFAOYSA-L 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000002753 trypsin inhibitor Substances 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
Abstract
Procedimiento para la extracción de un inhibidor de proteinasa a partir de tubérculos de patata que contienen el inhibidor proteinasa, que comprende las etapas de: (a) preparar una solución de extracción libre de alcohol mediante la adición de un ácido orgánico a agua en una concentración entre aproximadamente 0, 5 por ciento en peso y aproximadamente 2, 5 por ciento en peso y una sal en una cantidad para proporcionar entre aproximadamente 0, 3 y aproximadamente 5, 0 de sal común; (b) añadir el material vegetal a la solución de extracción en una relación en peso de entre aproximadamente 1:1 y aproximadamente 1:10 de solución de extracción respecto el material vegetal; y (c) pulverizar el material vegetal en la solución de extracción para reducir el tamaño de partícula medio del material vegetal hasta entre aproximadamente 100 micras y aproximadamente 1500 micras y generando así una suspensión que contiene una fracción sólida y una líquida; y (d) aislar y purificar el inhibidor de proteinasamediante centrifugación, clarificación, filtración y secado de dicha fracción líquida.Process for the extraction of a proteinase inhibitor from potato tubers containing the proteinase inhibitor, comprising the steps of: (a) preparing an alcohol-free extraction solution by adding an organic acid to water in a concentration between about 0.5 percent by weight and about 2.5 percent by weight and a salt in an amount to provide between about 0.3 and about 5.0 of common salt; (b) adding the plant material to the extraction solution in a weight ratio of between about 1: 1 and about 1:10 of extraction solution relative to the plant material; and (c) spraying the plant material in the extraction solution to reduce the average particle size of the plant material to between about 100 microns and about 1500 microns and thus generating a suspension containing a solid and a liquid fraction; and (d) isolate and purify the proteinase inhibitor through centrifugation, clarification, filtration and drying of said liquid fraction.
Description
Procedimiento que mejora la extracción de inhibidores de proteasa.Procedure that improves the extraction of protease inhibitors
La presente invención se refiere a un procedimiento para mejorar la extracción de un inhibidor de proteinasa, y más específicamente, a un procedimiento para mejorar el rendimiento y la pureza del Inhibidor II de Proteinasa (PI2) extraído de patatas enteras.The present invention relates to a procedure to improve the removal of an inhibitor of proteinase, and more specifically, to a procedure to improve the yield and purity of Proteinase Inhibitor II (PI2) extracted from whole potatoes.
La extracción, el aislamiento y la purificación de proteínas a partir de plantas es conocido en el campo de la bioquímica. En 1972, Melville y Ryan publicaron un procedimiento de preparación a gran escala para el aislamiento del inhibidor I de quimotripsina a partir de tubérculos de patata (Melville, J.C. y Ryan, C.A. Chymotrypsin inhibitor I from potatoes. J. Biological Chem., 247: 3445-3453, 1972). Según el método de Melville y Ryan, las patatas se cortaron con la piel intacta y se pusieron en remojo en una solución de hidrosulfito de sodio, se homogeneizaron, y se exprimieron a través de tela de nylon. El zumo resultante se ajustó a pH 3, se centrifugó a 1000 x g durante 15 minutos a 5ºF, y se recogió y fraccionó el sobrenadante con sulfato de amonio.The extraction, isolation and purification of proteins from plants is known in the field of biochemistry. In 1972, Melville and Ryan published a large-scale preparation procedure for the isolation of chymotrypsin inhibitor I from potato tubers (Melville, JC and Ryan, CA Chymotrypsin inhibitor I from potatoes. J. Biological Chem ., 247: 3445-3453, 1972). According to Melville and Ryan's method, the potatoes were cut with intact skin and soaked in a solution of sodium hydrosulfite, homogenized, and squeezed through nylon cloth. The resulting juice was adjusted to pH 3, centrifuged at 1000 xg for 15 minutes at 5 ° F, and the supernatant was collected and fractionated with ammonium sulfate.
La purificación se llevó a cabo mediante lavado en agua y tratamiento calorífico, se juntaron y liofilizaron los filtrados transparentes de las fracciones calentadas. El extracto crudo se obtuvo suspendiendo el polvo liofilizado en agua, sometiéndolo a diálisis contra agua durante 48 horas, y liofilizando el filtrado transparente resultante. A continuación, se centrifugó el extracto resuspendido y se cargó en una columna Sephadex G-75. Se juntaron las fracciones recogidas que contenían el inhibidor I, se evaporaron y se desalaron en una columna Sephadex G-25. Se determinó que el producto inhibidor resultante del filtrado en gel se purificó aproximadamente un 90% en la proteína inhibidora I mediante disociación en una columna Sephadex G-75 y desalado en una columna Sephadex G-25.Purification was carried out by washing in water and heat treatment, they were collected and lyophilized transparent filtration of heated fractions. The extract crude was obtained by suspending the lyophilized powder in water, subjecting it to dialysis against water for 48 hours, and lyophilizing the resulting clear filtrate. It was then centrifuged the resuspended extract and loaded on a Sephadex column G-75 The collected fractions that contained inhibitor I, evaporated and desalted in a Sephadex G-25 column. It was determined that the product inhibitor resulting from gel filtration was purified approximately 90% in inhibitor protein I by dissociation in a Sephadex G-75 column and desalted in a column Sephadex G-25.
El laboratorio de Ryan continuó publicando sobre el aislamiento y la caracterización del inhibidor II de proteinasa de manera parecida a como lo describió para el inhibidor I (Bryant, J., Green, T.R., Gurusaddaiah, T., Ryan, C.L. Proteinase inhibitor II from potatoes: Isolation and characterization of its protomer components. Biochemistry 15: 3418-3424, 1976). Bryant et al. diferenciaron los inhibidores proteinasa derivados de patata en dos grupos en función de su estabilidad a una temperatura de 80ºC durante 10 minutos. El inhibidor I de proteinasa (PI_{1}) está caracterizado como una proteína tetramérica compuesta de cuatro especies protoméricas hibridadas isoinhibidoras con un peso molecular de 39.000, mientras que PI2 está caracterizado como un inhibidor dimérico que comprende cuatro especies promotoras isoinhibidoras con un peso molecular de 21.000.Ryan's laboratory continued to publish on the isolation and characterization of proteinase inhibitor II in a similar way as he described for inhibitor I (Bryant, J., Green, TR, Gurusaddaiah, T., Ryan, CL Proteinase inhibitor II from potatoes: Isolation and characterization of its protomer components. Biochemistry 15: 3418-3424, 1976). Bryant et al . They differentiated potato-derived proteinase inhibitors into two groups based on their stability at a temperature of 80 ° C for 10 minutes. Proteinase inhibitor I (PI1) is characterized as a tetrameric protein composed of four isoinhibitory hybridized protomeric species with a molecular weight of 39,000, while PI2 is characterized as a dimeric inhibitor comprising four isoinhibitory promoter species with a molecular weight. of 21,000.
El documento WO 99/01474 describe la extracción y el aislamiento de las proteínas inhibidoras de proteinasa a partir de patatas. Las proteínas de los tubérculos de patata se extraen en forma soluble con un medio de extracción acuoso/alcohol, como por ejemplo diluyendo ácido fórmico y etanol 20%. El extracto alcohólico se calienta hasta una primera temperatura para desnaturalizar la mayor parte de las proteínas no deseadas y se enfría hasta una segunda temperatura para formar una fase de precipitado que contiene los restos de éstas y una fase soluble que contiene las proteínas inhibidoras de proteinasa estables frente al calor. Las proteínas inhibidoras de proteinasa estables frente al calor se precipitan desde la fase soluble mediante diálisis contra un medio de diálisis apropiado, como por ejemplo ácido fórmico diluido.WO 99/01474 describes the extraction and the isolation of proteinase inhibitor proteins to From potatoes Potato tuber proteins are extract in soluble form with an aqueous / alcohol extraction medium, such as diluting formic acid and 20% ethanol. The extract alcoholic is heated to a first temperature to denature most of the unwanted proteins and it cools to a second temperature to form a phase of precipitate that contains the remains of these and a soluble phase that contains stable proteinase inhibitor proteins against hot. Proteinase inhibitor proteins stable against heat is precipitated from the soluble phase by dialysis against an appropriate dialysis medium, such as formic acid diluted.
Recientemente, se ha asociado PI2 con el aumento
de la saciedad de individuos que han sido alimentados con una
composición nutricional en forma de bebida que contiene PI2. El
documento con número de publicación
US2002119915 describe
que, al ingerir una comida que comprendía una composición
nutricional en forma de bebida que contenía PI2, los individuos
mostraron una reducción importante del apetito durante hasta 3 horas
después de la comida. Además, se mejoró la sensación de saciedad, y
cada individuo del estudio perdió una media de 2 kg en un periodo
de 30 días sin experimentar los efectos secundarios adversos
asociados normalmente con los compuestos para la eliminación del
apetito. En relación a su mecanismo, se cree que, como inhibidor de
tripsina y de quimotripsina, cuando un individuo consume PI2
estimula la liberación de colecistoquinina endógena, un agente
supuestamente retroactivo conocido por que es efectivo en la
reducción de la necesidad de ingerir alimento.Recently, PI2 has been associated with the increase in satiety of individuals who have been fed a nutritional composition in the form of a drink containing PI2. The document with publication number
US2002119915 describes that, when eating a meal that comprised a nutritional composition in the form of a beverage containing PI2, the individuals showed a significant appetite reduction for up to 3 hours after the meal. In addition, the feeling of satiety was improved, and each individual in the study lost an average of 2 kg over a period of 30 days without experiencing the adverse side effects normally associated with appetite suppression compounds. In relation to its mechanism, it is believed that, as a trypsin and chymotrypsin inhibitor, when an individual consumes PI2 it stimulates the release of endogenous cholecystokinin, a supposedly retroactive agent known to be effective in reducing the need to eat food.
Los procedimientos existentes para la extracción de los inhibidores de proteinasa implican diversas etapas laboriosas y largas con lo que se pierde rendimiento y se reduce la pureza del inhibidor de proteinasa que se recupera. Además, los procedimientos más prometedores del estado de la técnica anterior se basan en la utilización de etanol en la solución de extracción que, en las concentraciones utilizadas, hace que la solución sea inflamable. Ninguno de los procedimientos del estado de la técnica anterior ha llegado a escala comercial. Por lo tanto, existe la necesidad de disponer de un procedimiento para la extracción a gran escala de PI2 de una manera efectiva y con costes apropiados que cumpla con los estándares industriales cualitativos y cuantitativos.Existing procedures for extraction of proteinase inhibitors involve various stages laborious and long with which performance is lost and the purity of the proteinase inhibitor that is recovered. In addition, the most promising prior art procedures are based on the use of ethanol in the extraction solution that, at the concentrations used, it makes the solution flammable. None of the prior art procedures Previous has reached commercial scale. Therefore, there is the need for a large extraction procedure scale of PI2 in an effective way and with appropriate costs that comply with qualitative industrial standards and Quantitative
Los tubérculos de patata que contienen un inhibidor de proteinasa de interés se combinan con una solución de un ácido orgánico y una sal. El material tuberculoso de patata se pulveriza, formando una suspensión en la solución de ácido y sal, para reducir el tamaño de partícula e incrementar el área superficial de las partículas para mejorar la eficiencia de la extracción. Se ha elegido un procedimiento de suspensión para reducir el tamaño de partícula sin desnaturalizar el inhibidor proteinasa de interés por causa de efectos locales de calentamiento. La solución de ácido y sal mejora la extracción del inhibidor de proteinasa a partir del material tuberculoso de patata suspendido y lo protege contra la degradación provocada por otros compuestos que pueden liberarse de las células vegetales lisadas. Una vez extraído en la solución, el inhibidor proteinasa se aísla y se purifica mediante centrifugación, clarificación, filtración y secado de la solución del extracto. El ácido y la sal se eliminan durante la etapa de filtración de manera que no se adultere el producto inhibidor de proteinasa purificado.Potato tubers that contain a Proteinase inhibitor of interest are combined with a solution of An organic acid and a salt. The potato tuber material is spray, forming a suspension in the acid and salt solution, to reduce the particle size and increase the area surface of the particles to improve the efficiency of the extraction. A suspension procedure has been chosen for reduce particle size without denaturing the inhibitor Proteinase of interest due to local effects of heating. The acid and salt solution improves the extraction of proteinase inhibitor from potato tuber material suspended and protects against degradation caused by others compounds that can be released from lysed plant cells. Once extracted in the solution, the proteinase inhibitor is isolated and it is purified by centrifugation, clarification, filtration and drying of the extract solution. The acid and salt are removed during the filtration stage so that the purified proteinase inhibitor product.
En una realización preferida, el inhibidor proteinasa II (PI_{2}) se extrae a partir de los tubérculos de patata enteros. Los ácidos orgánicos efectivos para el procedimiento incluyen ácido acético, ascórbico, cítrico y fórmico. El ácido fórmico resultó ser el que rindió el producto final PI2 de mayor grado de pureza y rendimiento. El contenido de ácido fórmico de la solución se ajusta al intervalo de 0,5% a 2,5% p/p, preferiblemente con un contenido de aproximadamente 1,5%. Se añade cloruro de sodio a la solución de extracción para incrementar la solubilidad de las proteínas de patata. Las concentraciones de cloruro de sodio utilizadas están entre 1 N y 3 N, preferiblemente una concentración de aproximadamente 1,5 N. La solución se añade a las patatas en una relación en peso de entre 1:1 y 1:10, preferiblemente una relación de 1:2,5 de solución de extracción:patata p/p, respectivamente.In a preferred embodiment, the inhibitor Proteinase II (PI2) is extracted from the tubers of whole potatoes Organic acids effective for the procedure they include acetic, ascorbic, citric and formic acid. Acid formic turned out to be the one that yielded the final PI2 final product degree of purity and performance. The formic acid content of the solution fits in the range of 0.5% to 2.5% w / w, preferably with a content of approximately 1.5%. Sodium Chloride is added to the extraction solution to increase the solubility of the potato proteins Sodium chloride concentrations used are between 1 N and 3 N, preferably a concentration of approximately 1.5 N. The solution is added to the potatoes in a weight ratio between 1: 1 and 1:10, preferably a ratio of 1: 2.5 extraction solution: potato p / p, respectively.
La pulverización se lleva a cabo mediante molido. El tamaño de partícula estará comprendido en el intervalo de 100 a 1500 \mum. En este intervalo, los rendimientos del producto se incrementaron y las características de fluidez de la suspensión fueron aceptables. Al disminuir el tamaño de partícula por debajo de 100 \mum, la recuperación de PI2 fue inferior y no mejoraron las características de fluidez. El molido durante un periodo mayor de tiempo también llevó a un rendimiento inferior de PI2, se cree que a causa de un incremento de la temperatura y de la liberación de proteasas no deseadas que hacen disminuir el rendimiento de PI2. El ácido fórmico y el cloruro de sodio se eliminan eficientemente durante la etapa de filtración.Spraying is carried out by ground. The particle size will be in the range from 100 to 1500 µm. In this interval, the yields of product increased and the flow characteristics of the suspension were acceptable. By decreasing particle size below 100 µm, the recovery of PI2 was lower and not improved fluidity characteristics. Grinding for a longer period of time also led to lower performance of PI2, it is believed that because of an increase in temperature and release of unwanted proteases that decrease the PI2 performance. Formic acid and sodium chloride are efficiently removed during the filtration stage.
Un objeto de la presente invención es proporcionar un procedimiento mejorado para la extracción de inhibidores de proteinasa de los tubérculos de patata.An object of the present invention is provide an improved procedure for the extraction of Proteinase inhibitors of potato tubers.
Otro objeto de la invención es proporcionar un procedimiento mejorado para la extracción de inhibidor II de proteinasa de tubérculos de patata sin utilizar etanol en la solución de extracción.Another object of the invention is to provide a improved procedure for the removal of inhibitor II from potato tuber proteinase without using ethanol in the extraction solution.
Otro objeto de la invención es proporcionar un procedimiento mejorado para la extracción de inhibidor II de proteinasa de tubérculos de patata, eficiente y rentable a escala comercial.Another object of the invention is to provide a improved procedure for the removal of inhibitor II from potato tuber proteinase, efficient and cost effective at scale commercial.
La extracción y aislamiento de PI2 a partir de patatas comienza con la adición de un ácido orgánico a las patatas de partida, preferiblemente ácido fórmico, y una sal, preferiblemente cloruro de sodio. La mezcla se pulveriza para reducir el tamaño de las partículas de patata y extraer proteínas solubles. Se utiliza centrifugación para eliminar sólidos y la fracción sólida se calienta a una temperatura suficiente para desnaturalizar muchas de las proteínas no deseadas, pero no el PI2. A continuación la solución se centrifuga de nuevo para eliminar las proteínas insolubles desnaturalizadas y se microfiltra la fracción líquida para eliminar las partículas relativamente grandes. La ultrafiltración se utiliza para eliminar el ácido orgánico y la sal y para purificar adicionalmente el PI2 en el concentrado.The extraction and isolation of PI2 from Potatoes begins with the addition of an organic acid to potatoes starting, preferably formic acid, and a salt, preferably sodium chloride. The mixture is sprayed to reduce the size of potato particles and extract proteins soluble. Centrifugation is used to remove solids and the solid fraction is heated to a temperature sufficient to denature many of the unwanted proteins, but not the PI2. The solution is then centrifuged again to remove the denatured insoluble proteins and the fraction is microfiltered liquid to remove relatively large particles. The Ultrafiltration is used to remove organic acid and salt and to further purify the PI2 in the concentrate.
Como intento para maximizar el rendimiento, minimizar las impurezas, minimizar el coste, y conseguir que fuera comercializable, se desarrolló un procedimiento para la extracción de PI2 a partir de patatas enteras. La solución de extracción se ensayó en base a la capacidad del procedimiento para solubilizar el PI2, proteger el PI2 de la degradación, y maximizar el PI2 total extraído de los componentes insolubles de la patata, a la vez que se minimiza la cantidad de proteínas cosolubilizadas. La solución de extracción presenta la solubilidad y estabilidad de PI2 en medio ácido y a temperaturas elevadas. Para este objetivo se ha encontrado apropiada una solución de extracción que contiene cloruro de sodio y ácido fórmico. A causa de los costes, se minimizó la relación utilizada de solución de extracción respecto al producto de partida a extraer, y a la vez se consiguió el rendimiento máximo de PI2 por kilogramo de tubérculos de patata de partida.As I try to maximize performance, minimize impurities, minimize cost, and get it out marketable, a procedure for extraction was developed of PI2 from whole potatoes. The extraction solution is tested based on the ability of the procedure to solubilize the PI2, protect PI2 from degradation, and maximize total PI2 extracted from the insoluble components of the potato, while the amount of cosolubilized proteins is minimized. The solution of extraction presents the solubility and stability of PI2 in medium acid and at high temperatures. For this purpose it has been found appropriate an extraction solution containing sodium chloride and formic acid. Because of the costs, the relationship was minimized used extraction solution with respect to the starting product to extract, and at the same time the maximum performance of PI2 was achieved by kilogram of potato tubers starting.
Las cantidades de PI2, Kunitz e inhibidores
carboxipeptidasa se midieron utilizando HPLC de fase inversa. Se
utilizó una columna Microsorb C-18 (4,6 mm x 250 mm,
partículas de 5 \mum con 300 Angstroms de tamaño de poro; Varian
Analytical Instruments). Se prepararon dos disolventes para la fase
móvil, el disolvente A compuesto de 800 g de H_{2}O desionizada,
150 g de acetonitrilo, y 0,95 de ácido trifluoroacético, y el
disolvente B compuesto de 850 g de acetonitrilo y 0,85 de ácido
trifluoroacético. Aproximadamente 50 mg de la muestra se añadió a
100 ml del disolvente A. La muestra se mezcló en un vortex durante
30 segundos y a continuación se centrifugó a 10.000 revoluciones
durante 10 minutos. El sobrenadante se recogió para someterlo a un
análisis RP-HPLC. Se inyectaron en la columna 100
\mul de la muestra, con la bomba ajustada a 2500 psig, y una
temperatura de 30,0ºC. Los otros caudales, tiempos, y
com-
posiciones de los disolventes se ajustaron tal como se
indica en la Tabla 1. El diodo del detector se ajustó a 220 nm.The amounts of PI2, Kunitz and carboxypeptidase inhibitors were measured using reverse phase HPLC. A Microsorb C-18 column (4.6 mm x 250 mm, 5 µm particles with 300 pore size Angstroms; Varian Analytical Instruments) was used. Two solvents were prepared for the mobile phase, solvent A composed of 800 g of deionized H2O, 150 g of acetonitrile, and 0.95 of trifluoroacetic acid, and solvent B composed of 850 g of acetonitrile and 0, 85 of trifluoroacetic acid. Approximately 50 mg of the sample was added to 100 ml of solvent A. The sample was mixed in a vortex for 30 seconds and then centrifuged at 10,000 revolutions for 10 minutes. The supernatant was collected for an RP-HPLC analysis. 100 µl of the sample was injected into the column, with the pump set at 2500 psig, and a temperature of 30.0 ° C. The other flows, times, and com-
solvent positions were adjusted as indicated in Table 1. The detector diode was adjusted to 220 nm.
Se preparó un patrón externo para construir una curva estándar para la calibración de la columna. Se disolvieron 5 mg de BSA en 10 ml de disolvente A. Se inyectaron cuatro volúmenes en la columna, es decir, 25, 50, 75 y 100 \mul. Con los resultados se generó una curva de calibración.An external pattern was prepared to build a standard curve for column calibration. 5 were dissolved mg of BSA in 10 ml of solvent A. Four volumes were injected in the column, that is, 25, 50, 75 and 100 µl. With the results A calibration curve was generated.
500 gramos de tubérculos de patata se sometieron a una extracción con 213 ml de una solución de ácido fórmico al 1% en un mezclador Waring durante 2,5 minutos. La suspensión se centrifugó a 10.000 rpm durante 40 minutos. Se decantó el líquido y se filtró a través de papel de filtro Whatman #4, rindiendo 486 g de extracto clarificado. En cada uno de 6 erlenmeyers con barras magnéticas de agitación se vertieron 50 gramos de este extracto clarificado. En cada matraz se añadió la cantidad de NaCl correspondiente según la Tabla 2 y se agitó hasta que se disolvió la sal. Los matraces se calentaron sobre una placa calefactora con agitación hasta que la temperatura del extracto alcanzó 70ºC. Después de dejarlos enfriar hasta temperatura ambiente, las soluciones se centrifugaron a 12.000 rpm durante 5 minutos y a continuación se analizaron utilizando el método de HPLC de fase inversa descrito arriba. El nivel de PI2 conseguido se calculó mediante la integración del área del pico PI2. El volumen de inyección fue 100 \mul y se utilizó la siguiente ecuación para equiparar las áreas de los picos a niveles de proteínas:500 grams of potato tubers were subjected to an extraction with 213 ml of a 1% formic acid solution in a Waring mixer for 2.5 minutes. The suspension is centrifuged at 10,000 rpm for 40 minutes. The liquid was decanted and filtered through Whatman # 4 filter paper, yielding 486 g of clarified extract In each of 6 erlenmeyers with bars Stirring magnetic 50 grams of this extract was poured clarified In each flask the amount of NaCl was added corresponding according to Table 2 and stirred until dissolved the salt. The flasks were heated on a heating plate with stirring until the temperature of the extract reached 70 ° C. After allowing them to cool to room temperature, the solutions were centrifuged at 12,000 rpm for 5 minutes and at They were then analyzed using the phase HPLC method reverse described above. The level of PI2 achieved was calculated by integrating the area of the PI2 peak. The volume of injection was 100 µl and the following equation was used to match the areas of the peaks to protein levels:
Proteína (mg/ml) = [(Área del pico/4) x 8,17 x 10^{-5}] + 0,0338Protein (mg / ml) = [(Peak area / 4) x 8.17 x 10-5] + 0.0338
Al extracto clarificado de patata se le
añadieron diferentes cantidades de cloruro de sodio, a continuación
se calentó a 70ºC durante 10 minutos. Después de enfriarlas hasta
temperatura ambiente, se analizó la elución de proteínas PI2
de
las soluciones mediante el método de HPLC para la
cuantificación de PI2. Los resultados se muestran en la Tabla 2.Different amounts of sodium chloride were added to the clarified potato extract, then heated at 70 ° C for 10 minutes. After cooling to room temperature, the elution of PI2 proteins from the
the solutions using the HPLC method for the quantification of PI2. Results are shown in table 2.
Para establecer la eliminación de las impurezas Kunitz del extracto de patata, que según se ha descrito disminuyen la efectividad de PI2 para incrementar la saciedad, se utilizó el método de HPLC de fase inversa sobre un patrón de Kunitz comercial provisto por Sigma. Un cromatograma del patrón de Kunitz reveló que el mayor pico de las impurezas Kunitz aparece aproximadamente a los 25 minutos. Otro inhibidor que se sabe presente en la patata es el inhibidor de carboxipeptidasa. Se utilizó el método de HPLC de fase inversa sobre un patrón de carboxipeptidasa comercial provisto por Sigma. El cromatograma del patrón de carboxipeptidasa reveló que los mayores picos de las impurezas carboxipeptidasa son un doblete que aparece aproximadamente a los 17 minutos. A niveles de 0,3 N de cloruro de sodio y superiores, el nivel de proteína posterior al tratamiento calorífico se mantiene relativamente constante. La cantidad de PI2 se mantiene relativamente constante en todos los ensayos, indicando que a 70ºC PI2 no precipita a niveles salinos hasta 0,5 N. Para llegar al nivel de NaCl necesario en la fase de tratamiento térmico es necesario utilizar un extractante con aproximadamente 2 veces la concentración final de sal. Así, en la solución de extracción es preferible un nivel de sal de al menos 0,3 N durante el tratamiento térmico a 70ºC para asegurar la eliminación eficaz de las proteínas de tipo Kunitz. La pureza del producto final de PI2 puede mejorarse con cantidades mayores de cloruro de sodio.To establish the removal of impurities Kunitz of potato extract, which as described decreases the effectiveness of PI2 to increase satiety, the reverse phase HPLC method on a commercial Kunitz pattern provided by Sigma. A chromatogram of Kunitz's pattern revealed that the biggest peak of Kunitz impurities appears approximately at 25 minutes. Another inhibitor known to be present in potatoes is the carboxypeptidase inhibitor. The phase HPLC method was used reverse on a commercial carboxypeptidase pattern provided by Sigma. The chromatogram of the carboxypeptidase pattern revealed that the major peaks of the carboxypeptidase impurities are a doublet that It appears at approximately 17 minutes. At 0.3 N levels of sodium chloride and higher, the level of protein after Heat treatment remains relatively constant. The amount of PI2 remains relatively constant in all tests, indicating that at 70 ° C PI2 does not precipitate at saline levels up to 0.5 N. To reach the level of NaCl required in the phase of heat treatment it is necessary to use an extractant with about 2 times the final salt concentration. So, in the Extraction solution a salt level of at least 0.3 is preferable N during heat treatment at 70 ° C to ensure removal Effective of Kunitz type proteins. The purity of the final product of PI2 can be improved with larger amounts of sodium.
Se realizó un estudio de optimización para determinar el contenido apropiado de NaCl y el contenido apropiado de ácido fórmico de la solución de extracción. La formulación ideal de la solución de extracción maximizaría la cantidad de PI2 liberada de la matriz de patata, a la vez que minimizaría la cantidad de contaminantes proteicos solubilizados. La liberación de PI2 se midió como rendimiento, normalizado para una composición de una solución de extracción de 1,0 N en NaCl. Se escogió ésta como base de normalización debido a la predicción previa de que el incremento del doble de NaCl por encima de 0,5 N se mostró efectivo para la eliminación de impurezas en el tratamiento térmico. Para la optimización, se midió la pureza proteica de PI2 y se comparó empíricamente con los rendimientos normalizados de extracción. Se examinaron las soluciones que contenían concentraciones de NaCl de 0,0 N a 2,0 N. De manera similar, se optimizó el contenido en ácido fórmico de la solución de extracción. Se ensayaron contenidos de ácido fórmico entre el 0,0 por ciento y el 2,5 por ciento.An optimization study was conducted to determine the appropriate NaCl content and the appropriate content of formic acid from the extraction solution. The ideal formulation of the extraction solution would maximize the amount of PI2 released from the potato matrix, while minimizing the amount of solubilized protein contaminants. The release of PI2 was measured as yield, normalized for a composition of a 1.0 N extraction solution in NaCl. This one was chosen as normalization basis due to the previous prediction that the double NaCl increase above 0.5 N was effective for the removal of impurities in heat treatment. For the optimization, protein purity of PI2 was measured and compared empirically with standardized extraction yields. Be examined solutions containing NaCl concentrations of 0.0 N to 2.0 N. Similarly, the acid content was optimized Formic extraction solution. Contents of formic acid between 0.0 percent and 2.5 percent.
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Aunque se observó que las normalidades de 0,5 N y superiores de NaCl conducían a rendimientos altos, se seleccionó una normalidad de 1,0 N para maximizar tanto el rendimiento como la pureza.Although it was observed that normalities of 0.5 N and higher NaCl led to high yields, was selected a normal of 1.0 N to maximize both performance and purity.
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Los datos indican la utilización de un contenido de 1,5% de ácido fórmico en la solución de extracción. Aunque otras concentraciones de ácido fórmico ofrecen rendimientos similares, un contenido de 1,5% de ácido fórmico maximiza claramente la pureza.The data indicates the use of a content 1.5% formic acid in the extraction solution. Although others formic acid concentrations offer similar yields, a 1.5% formic acid content clearly maximizes purity.
Se realizó un ensayo para determinar el efecto de la utilización de diferentes cantidades de la solución de extracción que comprende 1,5% de ácido fórmico y 1,0 N NaCl en agua sobre el rendimiento. La proporción en peso de las patatas respecto a la solución de extracción se modificó de 1:1 a 1:10. Las proporciones utilizadas y los rendimientos observados se muestran en la Tabla 11.An assay was performed to determine the effect of using different amounts of the solution of extraction comprising 1.5% formic acid and 1.0 N NaCl in water About performance The proportion in weight of the potatoes with respect The extraction solution was modified from 1: 1 to 1:10. The proportions used and the observed yields are shown in Table 11
Aunque el mayor rendimiento se obtuvo con el mayor porcentaje de solución de extracción, la ganancia total en rendimiento es mínima por encima de la relación 0,4 hasta uno (1:2,5 p/p solución de extracción respecto patatas, respectivamente). Se ha seleccionado esta relación con el objetivo de minimizar el coste de la solución de extracción y los problemas de la manipulación de los materiales, tales como calentamiento, enfriamiento y evaporación.Although the highest performance was obtained with the highest percentage of extraction solution, the total gain in performance is minimal above the ratio 0.4 to one (1: 2.5 p / p extraction solution with respect to potatoes, respectively). Be You have selected this relationship with the objective of minimizing the cost of the extraction solution and the handling problems of materials, such as heating, cooling and evaporation.
Los datos dictaron la elección de 1,0 N de cloruro de sodio como la concentración preferida en la solución de extracción para el aislamiento de PI2. La utilización de 1,0 N de cloruro de sodio resulta en un rendimiento maximizado de PI2 en las condiciones ensayadas, y aunque otras concentraciones llevan a rendimientos similares, la pureza de la proteína PI2 conseguida con la utilización de 1,0 N de NaCl está maximizada a 1,0 N. Pueden conseguirse mayores purezas de PI2 mediante la utilización de menos cloruro de sodio, aunque eso representaría una reducción del rendimiento de PI2. Este nivel de cloruro de sodio también es apropiado para la eliminación de las impurezas de tipo Kunitz. De manera similar, los datos llevan a la selección de 1,5% de ácido fórmico como la concentración preferida para la extracción de PI2. Una solución de extracción que contiene 1,5% de ácido fórmico muestra un comportamiento antimicrobiano y antiproteolítico beneficioso. El rendimiento de PI2 se maximiza en las condiciones ensayadas con una solución de extracción con un contenido de 1,5% en ácido fórmico, y esta concentración también proporciona la pureza más alta en PI2/Kunitz de las formulaciones con rendimientos comparables. No hay un incremento significativo en el rendimiento total cuando se crea una suspensión que está compuesta de un porcentaje mayor al 30% en peso de solución de extracción. Una suspensión del treinta por ciento en solución de extracción es aproximadamente equivalente a una parte de solución de extracción y una parte y media de material de partida (1:2,5 relación solvente:sólido).The data dictated the choice of 1.0 N of sodium chloride as the preferred concentration in the solution of extraction for the isolation of PI2. The use of 1.0 N of sodium chloride results in maximized yield of PI2 in the conditions tested, and although other concentrations lead to similar yields, the purity of the PI2 protein achieved with 1.0 N NaCl utilization is maximized to 1.0 N. They can achieve higher purities of PI2 by using less sodium chloride, although that would represent a reduction in PI2 performance. This level of sodium chloride is also appropriate for the removal of impurities of Kunitz type. From similarly, the data leads to the selection of 1.5% acid formic as the preferred concentration for the extraction of PI2. An extraction solution containing 1.5% formic acid shows an antimicrobial and antiproteolytic behavior beneficial. The performance of PI2 is maximized in the conditions tested with an extraction solution with a 1.5% content in formic acid, and this concentration also provides purity highest in PI2 / Kunitz of formulations with yields comparable. There is no significant increase in performance total when creating a suspension that is composed of a percentage greater than 30% by weight of extraction solution. A Thirty percent suspension in extraction solution is approximately equivalent to a part of extraction solution and a part and a half of starting material (1: 2.5 ratio solvent: solid).
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| US7829277B2 (en) | 2004-03-01 | 2010-11-09 | The Regents Of The University Of California | Methods for identifying compounds that suppress chemically-induced carcinogenesis |
| US20060035827A1 (en) * | 2004-06-24 | 2006-02-16 | Green Gary M | Compositions and methods for the treatment or prevention of gallbladder disease |
| US20070148267A1 (en) * | 2005-12-15 | 2007-06-28 | Svyatoslav Komarnytskyy | Method for producing plant extracts enriched with protease inhibitors for regulation of appetite and food intake in mammals |
| DE102007012439A1 (en) | 2007-03-15 | 2008-09-18 | Emsland-Stärke GmbH | Process for obtaining plant proteins and / or peptides, proteins and / or peptides produced therefrom and use thereof |
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| EP0699393B1 (en) * | 1994-09-03 | 1998-12-02 | Societe Des Produits Nestle S.A. | Process for preparing instant black tea |
| AU738102B2 (en) * | 1997-07-02 | 2001-09-06 | Washington State University Research Foundation | Methods for the isolation of proteinase inhibitor proteins from potato tubers |
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2001
- 2001-07-06 US US09/900,057 patent/US7371418B2/en not_active Expired - Fee Related
- 2001-07-06 US US09/900,555 patent/US6767566B2/en not_active Expired - Fee Related
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2002
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- 2002-06-20 JP JP2003509863A patent/JP4072122B2/en not_active Expired - Fee Related
- 2002-06-20 WO PCT/US2002/020115 patent/WO2003003838A1/en active IP Right Grant
- 2002-06-20 AT AT02737587T patent/ATE356146T1/en not_active IP Right Cessation
- 2002-06-20 EP EP02737587A patent/EP1414307B1/en not_active Expired - Lifetime
- 2002-06-20 DE DE60218694T patent/DE60218694T2/en not_active Expired - Lifetime
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| JP2004531582A (en) | 2004-10-14 |
| DE60218694T2 (en) | 2007-12-06 |
| WO2003003838A1 (en) | 2003-01-16 |
| US7371418B2 (en) | 2008-05-13 |
| US20030092152A1 (en) | 2003-05-15 |
| DE60218694D1 (en) | 2007-04-19 |
| EP1414307A1 (en) | 2004-05-06 |
| US20050037474A9 (en) | 2005-02-17 |
| EP1414307A4 (en) | 2004-12-15 |
| US6767566B2 (en) | 2004-07-27 |
| EP1414307B1 (en) | 2007-03-07 |
| US20030092150A1 (en) | 2003-05-15 |
| JP4072122B2 (en) | 2008-04-09 |
| ATE356146T1 (en) | 2007-03-15 |
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